Tissue culture allows the study in vitro of animal cell behavior in an investigator-controlled physiochemical environment. However, cellular morphology and metabolic activity of cultured cells are affected by the composition and architecture of the substrate on which they are grown. Presumably cultured cells function best (i.e. proliferate and/or perform their natural in vivo functions) when cultured on substrates that closely mimic their natural environment.
The interaction of cells with their extracellular matrix (ECM) in both in vivo and in vitro environments plays a crucial role in the organization, homeostasis, and function of all tissues and organs. Continuous communication between cells and the surrounding matrix environment orchestrates critical processes such as the acquisition and maintenance of differentiated phenotypes during embryogenesis, the development of form (morphogenesis), angiogenesis, wound healing, and even tumor metastasis. The cell and its ECM are said to exist in a state of "dynamic reciprocity". Both biochemical and biophysical signals from the ECM modulate fundamental cellular activities including adhesion, migration, proliferation, differential gene expression, and programmed cell death.
In turn, the cell can modify its ECM environment by modulating synthesis and degradation of specific matrix components. The realization of the significance of cell-ECM communication has led to a renewed interest in characterizing ECM constituents and the basic mechanisms of cell-ECM interaction.
Currently, studies conducted in vitro for analyzing cellular function are limited by the availability of cell growth substrates that present the appropriate physiological environment for proliferation and function/growth development of the cultured cells. To provide an in vitro cell culture environment which would more closely mimic cell-ECM interaction in vivo, purified ECM components such as collagen, fibronectin, laminin, glycosaminoglycans (e.g., hyaluronic acid, heparan sulfate) have been used to prepare artificial substrata for augmentation of cell adhesion, growth, and morphology. Three-dimensional (3D) culture matrices also have been fashioned from purified ECM components, specifically fibrin clots and collagen gels. Investigations with these matrices have demonstrated the importance of 3D architecture in the establishment of a tissue-like histology.
Complex scaffolds representing combinations of ECM components in a natural or processed form are commercially available. For example, Becton Dickinson currently offers two such products: Human Extracellular Matrix, and MATRIGEL.RTM. Basement Membrane Matrix. Human Extracellular Matrix is a chromatographically partially purified matrix extract derived from human placenta and comprises laminin, collagen IV, and heparin sulfate proteoglycan. (Kleinman, H K et al., U.S. Pat. No. 4,829,000 (1989).) MATRIGEL.RTM. is a soluble basement membrane extract of the Engelbreth-Holm-Swarm (EHS) tumor, gelled to form a reconstituted basement membrane. Both of these basement membrane extracellular matrix products require costly biochemical isolation, purification, and synthesis techniques and thus production costs are high.
Additional basement membrane matrices utilized as cell culture substrates include allogeneic and xenogeneic compositions prepared from lens capsule, liver, amnion, and chorioallantoic membranes. Although these substrata allow the study of cell growth and differentiation in a more physiologically relevant system, their use has been limited by availability and amenability to disinfection, sterilization, and manufacturing processes.
The present invention is directed to the preparation of a collagenous gel matrix derived from the interstitial extracellular matrix of warm-blooded vertebrate tissues. The predominant collagen types present such matrices are collagen I, III and V. The matrices for use in accordance with the present invention are derived from tissues comprising highly conserved collagens, glycoproteins, proteoglycans, and glycosaminoglycans in their natural configuration and natural concentration. One extracellular collagenous matrix for use in this invention is derived from submucosal tissue of a warm-blooded vertebrate.
Submucosal tissue harvested from warm-blooded vertebrates is a collagenous matrix that has shown great promise as a unique graft material for inducing the repair of damaged or diseased tissues in vivo, and for inducing the proliferation and differentiation of cell populations in vitro.
As a tissue graft, submucosal tissue undergoes remodeling and induces the growth of endogenous tissues upon implantation into a host. Numerous studies have shown that submucosal tissue is capable of inducing host tissue proliferation, remodeling and regeneration of tissue structures following implantation in a number of in vivo microenvironments, including lower urinary tract, body wall, tendon, ligament, bone, cardiovascular tissues and the central nervous system. It has been used successfully in vascular grafts, urinary bladder and hernia repair, replacement and repair of tendons and ligaments, and as a dermal graft. Upon implantation, cellular infiltration and a rapid neovascularization are observed and the submucosa extracellular matrix material is remodeled into host replacement tissue with site-specific structural and functional properties.
Vertebrate submucosa can be obtained from various sources, including intestinal tissue harvested from animals raised for meat production, including, for example, pigs, cattle and sheep or other warm-blooded vertebrates. The preparation and use of submucosa as a tissue graft composition is described in U.S. Pat. Nos. 4,902,508; 5,281,422; 5,275,826; 5,554,389 and other related U.S. patents. Submucosal tissue consists primarily of extracellular matrix material and is prepared by mechanically removing selected portions of the mucosa and the external muscle layers and then lysing resident cells with hypotonic washes. Preliminary biochemical analyses show that the composition of small intestinal submucosa is similar to that of other interstitial extracellular matrix structures, and consists of a complex array of collagen, proteoglycans, glycosaminoglycans, and glycoproteins. The major components commonly identified in extracellular matrix tissues similar to submucosal tissue include growth factors; the cell adhesion proteins, fibronectin, vitronectin, thrombospondin, and laminin; the structural components, collagen and elastin; and the proteoglycans, serglycin, versican, decorin, and perlecan.
Submucosa tissue can be used as a tissue graft construct, or as a cell culture substrate/supplement, in either its native solid form, as a fluidized comminuted form, or as an enzyme digested solubilized form. Furthermore, the solubilized forms of vertebrate submucosa can be gelled to form a semi-solid composition that can be implanted as a tissue graft construct or utilized as a cell culture substrate. As a tissue graft, the enzyme-digested, solubilized form can be injected or otherwise delivered to living tissues to augment, enhance or suppress the structure or function of said tissue. Furthermore, said enzyme-digested, solubilized form can be combined or modified with specific proteins, growth factors, drugs, plasmids, vectors, or other therapeutics agents for controlling the desired augmentation, enhancement or suppression of the recipient tissue function. Still further, said enzyme-digested, solubilized form can be combined with living primary or cultured cells prior to delivery to the living tissues, such combination providing further augmentation, enhancement of suppression of tissue structure or function. The submucosa gel form can also be used as a cell growth substrate, providing a relatively inexpensive cell culture growth substrate that promotes or induces growth and differentiation of cells cultured in vitro.